The long-term goal of this project is to define the mechanisms regulating motility of the first migratory cell type in the mammalian embryo, parietal endoderm (PE). The F9 teratocarcinoma system provides an in vitro model in which F9-derived embryoid bodies generate PE outgrowth when plated on an extracellular matrix (ECM)-coated substrate. Cells leave the embryoid body and migrate away as a sheet of PE. At the edge of the outgrowth, the outermost ring cells extend filopodia and lamellipodia and actively migrate away from the embryoid body, while remaining attached to the cells behind them. We have shown that focal adhesion turnover and lamellipodia formation by the outermost ring cells contribute to PE outgrowth. We also established a role for the small GTPase Rho and its downstream effector ROCK in maintaining robust focal adhesions and applying the brakes to PE migration. Time-lapse analysis led us to hypothesize a "push/pull" model for PE outgrowth in which the cells leaving the embryoid body spread and intercalate, pushing the outgrowth away from the embryoid body, while the outermost ring cells pull the outgrowth away from the embryoid body. Specific Aim 1. To define the role of Rho family GTPases in PE outgrowth. These studies will continue to examine the role of Rho and initiate analysis of the roles of Rac and Cdc42 using gain and loss of function approaches. We will identify major effectors and determine the subcellular localization of the active GTPases in migrating cells. Specific Aim 2. To determine if directed migration of PE is regulated by the PCP pathway. This aim tests the hypothesis that PE outgrowth is an example of directed migration using intercalation movements mediated by the PCP pathway. We will use gain and loss of function approaches. Specific Aim 3. To examine PE migration on a complex ECM and evaluate the role of integrins in modulating the mode of migration. The in vivo substrate for PE migration consists initially of fibronectin, and then Reichert's membrane, a complex matrix similar to the components of Matrigel. We will therefore examine PE outgrowth on Matrigel, plus and minus fibronectin, with a particular focus on examining the role of integrins in promoting directed versus random migration. [unreadable] [unreadable] [unreadable]